Programme Contents

Determination of the junctional space [Ca²⁺] set by ryanodine receptor Ca²⁺ leak in fast- and slow-twitch muscle fibres

The tubular (t-) system of skeletal muscle forms a junction with the sarcoplasmic reticulum (SR), with some 12nm between the membranes, at every sarcomere for the main purpose of conducting excitation-contraction coupling. In the resting muscle, [Ca²⁺] within the small volume bound by the junctional membranes will be determined by the leak of Ca²⁺ through the SR ryanodine receptors (RyRs), the Ca²⁺ handling ability of the t-system and diffusion of Ca²⁺ from the junctional space (js). The [Ca²⁺]_js is expected to be higher than the bulk cytoplasmic [Ca²⁺] ([Ca²⁺]_js) with a standing gradient set between the RyRs and SR Ca²⁺-pumps in the resting muscle. The value of [Ca²⁺]_js is unknown but has significant implications for signalling cascades initiating in this nanodomain. The value of [Ca²⁺]_js could also be expected to change under conditions of RyR mutation, which underlie a number of myopathies. Our aim was to develop a method to determine the [Ca²⁺]_js in fast- and slow-twitch muscle fibres. To do this we exploited the fact that t-system Ca²⁺ uptake activity will be set by [Ca²⁺]_js (and the t-system Ca²⁺ gradient) to determine [Ca²⁺]_js.

All experimental procedures were approved by The Animal Ethics Committee of The University of Queensland. Rats were euthanized by CO₂ asphyxiation and the extensor digitorum longus and soleus muscle were rapidly excised. Muscles were pinned down in a Petri dish above a layer of Sylgard under a layer of paraffin oil. Fibre bundles were isolated and a Ringer solution containing rhod-5N was applied to the fibres. Fibres were isolated and mechanically skinned and placed in a custom-built experimental chamber for imaging on an Olympus FV1000 confocal microscope.

Chronic depletion of [Ca²⁺]_SR with caffeine reduced [Ca²⁺]_t-sys to 0.1 mM via chronic activation of store-operated Ca²⁺ entry, providing a a consistent starting point for tracking t-system Ca²⁺ uptake. We then exposed Ca²⁺-depleted preparations to a solution containing either 50, 100, 200 or 800nM [Ca²⁺] in 50mM EGTA to allow observation of t-system Ca²⁺ uptake rates at known [Ca²⁺]_bulk. The t-system was subsequently depleted of Ca²⁺ to return [Ca²⁺]_t-sys to 0.1 mM and the cycle was repeated. Experiments were repeated in the presence of 1mM tetracaine to block RyR Ca²⁺ leak and allow [Ca²⁺]_js to equilibrate with [Ca²⁺]_bulk. Rhod-5N signals and [Ca²⁺]_t-sys were calibrated and t-system Ca²⁺ fluxes were derived. [Ca²⁺]_bulk and peak t-system Ca²⁺ fluxes were fitted by Hill curves. V_max was significantly depressed in slow- compared to fast-twitch fibres. The k_D of Hill curves fitted to data for both fibre types was right-shifted by tetracaine compared to the absence of tetracaine. It followed that at 100nM [Ca²⁺]_bulk, [Ca²⁺]_js was 165 and 220nM in slow and fast-twitch fibres, respectively. These results show that t-system Ca²⁺ fluxes can be used as a nanodomain sensor of RyR leak.